Chorismate- and Isochorismate Converting Enzymes: Versatile Catalysts Acting on an Cite This: Chem

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Chorismate- and isochorismate converting enzymes: versatile catalysts acting on an Cite this: Chem. Commun., 2021, 57, 2441 important metabolic node†

a b b Florian Hubrich, * Michael Mu¨ller and Jennifer N. Andexer *

Chorismate and isochorismate represent an important branching point connecting primary and secondary metabolism in bacteria, fungi, archaea and plants. Chorismate- and isochorismate-converting enzymes are potential targets for new bioactive compounds, as well as valuable biocatalysts for the in vivo and in vitro synthesis of fine chemicals. The diversity of the products of chorismate- and isochorismate-converting enzymes is reflected in the enzymatic three-dimensional structures and molecular mechanisms. Due to the high reactivity of chorismate and its derivatives, these enzymes have evolved to be accurately tailored to theirrespectivereaction;atthesametime,manyofthem exhibit a fascinating flexibility regarding side reactions and acceptance of alternative substrates. Here, we give an overview of the diﬀerent (sub)families Creative Commons Attribution-NonCommercial 3.0 Unported Licence. of chorismate- and isochorismate-converting enzymes, their molecular mechanisms, and three-dimensional structures. In addition, we highlight important results of mutagenetic approaches that generate a broader Received 12th December 2020, understanding of the influence of distinct active site residues for product formation and the conversion of Accepted 12th February 2021 one subfamily into another. Based on this, we discuss to what extent the recent advances in the field might DOI: 10.1039/d0cc08078k influence the general mechanistic understanding of chorismate- and isochorismate-converting enzymes. Recent discoveries of new chorismate-derived products and pathways, as well as biocatalytic conversions of rsc.li/chemcomm non-physiological substrates, highlight how this vast field is expected to continue developing in the future. This article is licensed under a

a ETH Zurich, Institute of Microbiology, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland. E-mail: [email protected] b Albert-Ludwigs-University Freiburg, Institute of Pharmaceutical Sciences, Albertstrasse 25, 79104 Freiburg, Germany. E-mail: [email protected] † Dedicated to Professors Heinz G. Floss and Eckhard Leistner for their inspiring research into chorismate-converting enzymes. Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM.

Florian Hubrich studied Michael Mu¨ller studied chemistry chemistry and biology at the at the University of Bonn. After University of Freiburg. receiving the diploma degree, he Following his Staatsexamen he began his PhD at the Ludwig- startedhisPhDinchemical Maximilians-Universita¨tinMunich biology with focus on under the guidance of Professor chorismatases supervised by Steglich, finishing in 1995. Jennifer Andexer and Michael Following a research exchange at Mu¨ller at the Institute of the University of Washington Pharmaceutical Sciences in working, he became the Group Freiburg and finished in 2014. Leader of Bioorganic Chemistry at In 2015 he moved to the Forschungszentrum Ju¨lich. Prof. Florian Hubrich Switzerland where he worked Michael Mu¨ller Mu¨ller qualified as a University for the BACHEM AG as project Lecturer for Organic and chemist and group leader for UHPLCanalysisofpeptides.In Bioorganic Chemistry in 2002 and was appointed the Professorship 2018 he returned to academia and joined Jo¨rn Piel’s lab as a for Pharmaceutical and Medicinal Chemistry at the Albert-Ludwigs- postdoctoral researcher at the ETH Zurich. Currently, he studies Universta¨t Freiburg in 2004. His research interests include the biosynthesis of peptide natural products from ribosomal chemoenzymatic synthesis, natural products, asymmetric and origin. diversity-oriented synthesisaswellassustainability.

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1. Introduction 1.1. Chorismate and isochorismate: highly reactive molecules with an enormous potential for diversity-oriented In 2001, Dosselaere and Vanderleyden described chorismate (1)asa product formation ‘‘metabolic node in action’’ in an outstanding review of the five most important families of chorismate-converting enzymes Chorismate (1) and its isomer isochorismate (2, 2-hydroxy-4- in microorganisms.1 At that time, eight products of immediate deoxychorismate) feature several chemical moieties attached to chorismate (1)andisochorismate(2)origin‡ were known, among its central cyclohexadiene system: a hydroxyl and a carboxyl them the amino derivatives 2-amino-2-deoxyisochorismate (3,ADIC) group as well as an enolpyruvyl ether. These moieties make and 4-amino-4-deoxychorismate (4,ADC),whichmaybecon- (iso)chorismate highly reactive under standard conditions and sidered equally versatile branching points, as discussed below challenging to handle. Notably, the reactivity of (iso)chorismate (Section 13).1–5 Within the last two decades, this number of products is a prerequisite for ‘diversity-oriented’ product formation in from a single metabolic substrate has increased to at least twelve enzymatic and non-enzymatic reactions, as studied intensively 14–20 (amino/iso)chorismate derivatives. In addition, the knowledge of the since the 1960s. structural basis, the molecular mechanisms of the enzymes 1.1.1. Non-enzymatic reactions of chorismate and involved, and the regulation of product formation has grown isochorismate. The non-enzymatic reactions from chorismate enormously. to prephenate (5) as well as 4-hydroxybenzoate (6, 4-HBA) are Here, we present an update on the structural basis and competitive with each other and formally pericyclic reactions underlying molecular mechanisms of the entire class of chor- (Fig. 1). The [3,3]-sigmatropic Claisen rearrangement from ismate- and isochorismate-converting enzymes.§ Rather chorismate to prephenate is favoured over the [1,5]- than providing an exhaustive compilation of original experi- sigmatropic rearrangement from chorismate to 4-HBA via ments, we aimed to prepare a general overview serving elimination of the enolpyruvyl side chain which ends up as as a starting point for research into the field and as a comple- pyruvate (7, see Scheme 1A and B). Analogously, the isomeric ment to a wealth of prior subfamily- and subtopic-specific products are obtained from isochorismate: the [3,3]- Creative Commons Attribution-NonCommercial 3.0 Unported Licence. reviews.6–13 sigmatropic Claisen rearrangement from isochorismate to iso- In addition, recent advances in the field and their potential prephenate (9) is similarly favoured over the [1,5]-sigmatropic influence on the understanding of chorismate-converting rearrangement from isochorismate to salicylate (8, Scheme 1C 16,18,19 enzymes in general are highlighted, followed by a brief overview and D). of novel chorismate-derived natural products and biocatalytic Data from experiments addressing the kinetic isotope eﬀect applications of chorismate-converting enzymes. via nuclear magnetic resonance (NMR) spectroscopy in combination with computational calculations indicate that both reactions proceed via a pseudo-diaxial chair-like transition state (TS), although the concerted mechanism is highly asynchro- This article is licensed under a nous (Scheme 1).18,19,21 Quantum mechanics and molecular mechanics calculations (QM/MM) support the pericyclic nature Following a diploma in Biology, of the reactions in solution. Nevertheless, the rate-limiting step

Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. Jennifer Andexer carried out her of the reactions, including their enzymatic counterparts, is still 22–26 doctoral research working on under discussion. Hilvert and co-workers determined that oxynitrilases at the Institute of the thermal stability of chorismate at 60 1C is higher than the 18,19,21 Molecular Enzyme Technology thermal stability of isochorismate. Comparable findings (University of Duesseldorf) were obtained by us with chorismate and isochorismate at supervised by Thorsten Eggert, 25 1C; however, this study further suggested that the half-life 27 Karl-Erich Jaeger and Martina time strongly depends on the purity of the compounds. The Pohl. In 2008, she moved to the molar enthalpy of the reaction from chorismate to isochorismate 1 labs of Joe Spencer, Finian Leeper in solution was determined as DrHm ¼ð0:81 0:27Þ kJ mol and Peter Leadlay (University of by the Goldberg group.28 Cambridge), studying biosynthetic 1.1.2. Enzymatic reactions of chorismate and isochorismate. Jennifer N. Andexer pathways of natural products. She These, if not all, non-enzymatic reactions of (iso)chorismate hasbeenheadoftheChemical in solution have enzymatic counterparts. The enzymatic Biology group (Institute of Pharmaceutical Sciences, University of reactions are at least 106-fold faster than the non-enzymatic Freiburg) since 2011 and was appointed Heisenberg Professor of reactions in solution with comparable molecular mechanisms Pharmaceutical and Medicinal Chemistry in 2020. She works on during product formation. This increase in the reaction rate enzyme selectivity, cofactor regeneration systems and cofactor is assumed to be caused by an entropic advantage of the analogues. enzymatic reaction caused by electrostatic networks assisting

‡ In other publications, para-aminobenzoate synthase is counted towards chorismate-converting enzymes, although these enzymes reactions do not act directly from chorismate (1) and isochorismate (2). § From here on named chorismate-converting enzymes to simplify reading and understanding of the text.

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Fig. 1 Chorismate (1), isochorismate (2), and (iso)chorismate derivatives. The colours indicate the diﬀerent families of chorismate-converting enzymes that catalyse the respective reaction: orange – MST (involved in the biosynthesis of menaquinone, siderophores and tryptophan) enzymes, red – SPR (catalysing solely pericyclic reactions) enzymes, green – isochorismatases, olive – chorismatases, blue – chorismate dehydratases, purple – SEPHCHC

Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. synthases, dashed lines – non-enzymatic reactions.

a product specific orientation of substrate, intermediates Starting from chorismate or isochorismate, important and/or transition states (TS) from the respective enzymatic primary and secondary metabolites such as ubiquinone (11), environment (see Sections 2–8).18,19,21,29,30 Moreover, a large menaquinone (12), siderophores [e.g., enterobactin (13)], number of additional enzymatic reactions have been described. folates (14), and the aromatic amino acids tryptophan (Trp, These (iso)chorismate-derived enzyme products are mainly 15), phenylalanine (Phe, 16), and tyrosine (Tyr, 17) are synthe- small, cyclic, often chiral and/or aromatic compounds, e.g. sised (Fig. 2).3,36 A wide range of bioactive compounds is (dihydro)benzoates, (iso)prephenate, and anthranilate (10). accessible via biosynthetic pathways branching from (iso)chor- Many of these have been used as building blocks for ismate or using (iso)chorismate derivatives as starter units for the chemoenzymatic synthesis of more complex compounds thiotemplate biosynthesis, e.g. of stravidin (18, antibiotic), which are challenging to produce via standard organic rapamycin (19, immunosuppressant), and unantimycins (20, synthesis.10,11,31–33 Examples of such biocatalytic applications anticancer agents), or for terpenoids such as merosterols (21, are given in Section 12. cytotoxic) (Fig. 2).37–40 As the shikimate and the corresponding branching biosynthetic pathways are not present in the Animalia kingdom including humans, these pathways and 1.1.3 Chorismate as a central branching point in metabolism. the enzymes involved are promising targets for the develop- In vivo, chorismate is the formal endpoint of the shikimate ment of selective drugs and pesticides.1,34 Nevertheless, there biosynthetic pathway and a central branching point connecting are hints that shikimate-like pathways might be present in this the primary and the secondary metabolism in bacteria, plants, phylum as summarised in a recent review on the natural and fungi.1,34,35 product potential of the animal world.41

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Scheme 1 Non-enzymatic reactions of chorismate and isochorismate, and molecular mechanisms of SPR enzymes, including near attack conformation (NAC) and transition state (TS). (A) [3,3]-Sigmatropic rearrangement of chorismate (1) to prephenate (5) (non-enzymatic and by chorismate mutases). (B) [1,5]-Sigmatropic rearrangement of chorismate (1) to 4-HBA (6) and pyruvate (7) (non-enzymatic and by chorismate lyases). (C) [3,3]-Sigmatropic rearrangement of isochorismate (2) to isoprephenate (9) (non-enzymatic). (D) [1,5]-Sigmatropic rearrangement of isochorismate (2) to salicylate (8) and pyruvate (7) (non-enzymatic and by isochorismate-pyruvate lyases). (E) Non-natural [1,5]-sigmatropic rearrangement of 4-amino-4-deoxychorismate (4) to pABA (24) and pyruvate (7) (by chorismate lyases).

2. A handful of enzymes access a cases, one subfamily with the same enzyme fold catalyses the plethora of products by a vast number formation of different products, while other examples are known where two subfamilies featuring different enzyme folds of molecular mechanisms provide the same product (Fig. 1 and Table 1). The structural Due to its role as a metabolic branching point, (iso)chorismate diversity of chorismate-converting enzymes (Fig. 3 and 4) is serves as a substrate for many different enzymes. Therefore, it echoed in the diversity of catalytic mechanisms (Schemes 2–7). is not surprising that non-enzymatic reactions and products of The majority of molecular mechanisms in chorismate- (iso)chorismate in solution have enzymatic counterparts. Fig. 2 converting enzymes are either pericyclic reactions or reactions shows a selection of products formed from (iso)chorismate. requiring acid–base catalysis. Nonetheless, several chorismate- Remarkably, structural studies on chorismate-converting converting enzymes are suggested to catalyse both reaction enzymes revealed that only a few different enzyme folds provide types in the same active site (Table 1). Pericyclic reactions access to this vast range of products (Fig. 3 and 4). In some are fascinating reactions in general; nevertheless, seemingly

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Fig. 2 Selected natural products and primary metabolites of (iso)chorismate origin: ubiquinone (11), menaquinone (12), enterobactin (13), folate (14), tryptophan (15), phenylalanine (16), tyrosine (17), stravidin (18), rapamycin (19), unantimycin A (20), merosterol A (21), chloramphenicol (22), cuevaene A(33), xanthomonadin I (34), brasilicardin (35). (Iso-)chorismate-derived moieties are red.

common acid–base reactions catalysed by chorismate- last two decades.7,21,29,43–47 Approaches using labelled sub- converting enzymes often follow sophisticated routes. One strates and/or solvents, rational site-directed mutagenesis, major factor determining product formation in chorismate- and inhibition studies are just a few examples of how open converting enzymes are electrostatic effects caused by amino questions were resolved.7,18,19,21,29,47–49 The ‘holy grail’ of these acid residues of the active site and the so-called secondary studies is often considered the conversion of the activity of a shell.42 These electrostatic effects contribute at various time chorismate-converting enzyme of one (sub)family into one of points to the reaction trajectory. They usually tightly lock the another subfamily. So far, most of these attempts were substrate in a pseudo-diaxial conformation that promotes unsuccessful, or accompanied by a drastic loss of activity. proper product formation by stabilising distinct transition In several cases, highly promiscuous enzymes were states or intermediates, and thus determine product specificity generated.47,49–51 and selectivity. Based on the structural knowledge of The following Sections 3–8 each focus on one group of chorismate-converting enzymes, a broader understanding of currently known chorismate-converting enzymes. Due to the product formation has been one main focus of studies for the diversity of three-dimensional structures, reactions and

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Fig. 3 Three-dimensional folds of chorismate-converting enzymes in complex with substrates, products, transition state analogues, or competitive inhibitors (grey). All structures are presented in their catalytic essential forms; for oligomers a single subunit is coloured analogously toFig.1: orange – MST enzymes, red – SPR enzymes, green – isochorismatases, olive – chorismatases, blue – chorismate dehydratases, purple – SEPHCHC synthases. (A) Homodimeric isochorismate-pyruvate lyase PchB with co-crystallised salicylate (8) and pyruvate (7) (PDB: 3REM). (B) Homodimeric chorismate mutase AroQ with co-crystallised 8-hydroxy-2-oxa-bicyclo[3.3.1]non-6-ene-3,5-dicarboxylate (PDB: 2FP2). (C) Monomeric chorismate lyase UbiC with co-crystallised 4-HBA (6) (PDB: 1TT8). (D) Monotrimeric chorismate mutase AroH with co-crystallised 8-hydroxy-2- oxa-bicyclo[3.3.1]non-6-ene-3,5-dicarboxylate (PDB: 3ZP4). (E) Monomeric chorismatase FkbO with co-crystallised 3-(2-carboxyethyl)benzoate (PDB: 4BPS). (F) Monomer of the homodimeric isochorismatase PhzD with co-crystallised ADIC (3) (PDB: 3R77). (G) Monomeric isochorismate synthase EntC with co-crystallised isochorismate (2) and magnesium ion (PDB: 3HWO). (H) Monomer of the homodimeric chorismate dehydratase MqnA with co-crystallised 3-HBA (32) (PDB: 6O9A). (I) Dimer of the homotetrameric SEPHCHC synthase MenD with co-crystallised magnesium ion, thiamine diphosphate and isochorismate (2)(PDB:5ESO).

mechanisms, there is not a single clear-cut classification Enzymes involved in the biosynthesis of Menaquinone, scheme. Taking into account the advances in the field over Siderophores, and Tryptophan (MST-enzymes, Section 4) the last decade(s),1–3,7,8 we decided on the following classifica- (Iso-)chorismate hydrolases (isochorismatases) and type-I tion of chorismate-converting enzyme families: chorismatases (Section 5) Enzymes Solely catalysing Pericyclic Reactions (SPR- SEPHCHC synthases (Section 6) enzymes, Section 3) Chorismate dehydratases (Section 7)

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Fig. 4 Close-up of active sites from structures shown in Fig. 3. (A) Isochorismate-pyruvate lyase PchB with co-crystallised salicylate (8)andpyruvate(7)(PDB: 3REM). (B) Chorismate mutase AroQ with co-crystallised 8-hydroxy-2-oxa-bicyclo[3.3.1]non-6-ene-3,5-dicarboxylate (PDB: 2FP2). (C) Chorismate lyase UbiC with co-crystallised 4-HBA (6)(PDB:1TT8).(D)ChorismatemutaseAroHwithco-crystallised8-hydroxy-2-oxa-bicyclo[3.3.1]non-6-ene-3,5-dicarboxylate (PDB: 3ZP4). (E) Chorismatase FkbO with co-crystallised 3-(2-carboxyethyl)benzoate (PDB: 4BPS). (F) Isochorismatase PhzD with co-crystallised ADIC (3)(PDB: 3R77). (G) Isochorismate synthase EntC with co-crystallised isochorismate (2) and magnesium ion (PDB: 3HWO). (H) Chorismate dehydratase MqnA with co- crystallised 3-HBA (32) (PDB: 6O9A). (I) SEPHCHC synthase MenD with co-crystallised magnesium ion, thiamine diphosphate and isochorismate (2)(PDB: 5ESO). Amino acid residues located on diﬀerent quarternary structure elements are shown in colour and grey.

Chorismatases (Section 8) 3. Enzymes solely catalysing pericyclic This classification is based on the type of reaction, and reactions: (iso)chorismate mutases and in many cases goes along with similar three-dimensional lyases structures and molecular mechanisms. An exception are SPR enzymes, as this group contains subfamilies with Although the number of known enzyme families catalysing diﬀerent enzyme folds. Chorismatases represent another pericyclic reactions has increased over the last decade, this exception, as they are characterised by a single enzyme type of enzymatic reaction remains uncommon.52 (Iso)choris- fold (Section 8) but perform fundamentally diﬀerent mate mutases, chorismate lyases, and isochorismate-pyruvate product formation after a shared initial reaction step lyases solely catalyse pericyclic reactions; in the following, these (Sections 5 and 8). are referred to as the SPR enzyme family (Solely catalysing

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Table 1 Overview of families, subfamilies, enzyme folds, molecular mechanisms, and products of chorismate-converting enzymes

Classification Example Name (accession EC Enzyme Molecular Family Subfamily number) Organism number fold mechanism(s) Product(s) SPR enzymes AroH-chorismate AroH (P19080) Bacillus subtilis 5.4.99.5 AroH-fold [3,3]-Sigmatropic Prephenate (5) mutases rearrangement AroQ-chorismate AroQ (Q9HU05) Escherichia coli 5.4.99.5 AroQ-fold [3,3]-Sigmatropic Prephenate (5) mutases rearrangement Isochorismate- PchB (Q51507) Pseudomonas 4.2.99.21 [1,5]-Sigmatropic Salicylate (8) + pyruvate pyruvate lyases aeruginosa rearrangement (7) Chorismate lyases UbiC (P26602) Escherichia coli 4.1.3.40 UbiC-fold [1,5]-Sigmatropic 4-HBA (6) + pyruvate (7) Chorismate- rearrangement pyruvate lyases

MST enzymes Isochorismate EntC (P0AEJ2) Escherichia coli 5.4.4.2 MST-fold Nucleophilic sub- Isochorismate (2) synthases stitution (SN2) Salicylate synthases MbtI (P9WFX1) Mycobacterium 5.4.4.2 Nucleophilic sub- Salicylate (8) + pyruvate tuberculosis stitution (SN2) (7) 4.2.99.21 [1,5]-Sigmatropic rearrangement 2-Amino-2- PhzE (G3XCK6) Pseudomonas 2.6.1.86 Nucleophilic sub- 2-Amino-2- deoxyisochorismate aeruginosa stitution (SN2) deoxyisochorismate (ADIC) synthases (ADIC) (3) Anthranilate TrpE (P00897) Serratia 4.1.3.27 Nucleophilic sub- Anthranilate (10)+ synthase marcescens stitution (SN2) pyruvate (7) [1,5]-Sigmatropic rearrangement 4-Amino-4- PabA (P06193) Stenotrophomonas 2.6.1.85 2 nucleophilic 4-Amino-4- Creative Commons Attribution-NonCommercial 3.0 Unported Licence. deoxychorismate maltophilia substitution (SN2) deoxychorismate (ADC) (ADC) synthases (4)

Isochorismatases Isochorismatases EntB (P0ADI4) Escherichia coli 3.3.2.1 IHL-fold Vinyl ether 2,3-trans-CHD (25)/2,3- hydrolysis trans-CHA (26)/3,4-trans- CHD (25)/3,4-trans-CHA (28) + pyruvate (7)

SEPHCHC SEPHCHC MenD (P17109) Escherichia coli 2.2.1.9 ThDP-fold 1,4-Conjugate SEPHCHC (29) synthases synthases addition This article is licensed under a Chorismate Chorismate MqnA (Q5SK49) Deinococcus 4.2.1.151 MqnA-fold Elimination (E1cb) Enoyl benzoate (31) dehydratases dehydratases radiodurans

Chorismatases Type-I FkbO (Q9KID9) Streptomyces 3.3.2.13 Toblerone- Vinyl ether 3,4-trans-CHD (27)+ chorismatases hygroscopicus fold hydrolysis pyruvate (7) Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. subsp. ascomyceticus Type-II Hyg5 (O30478) Streptomyces 4.1.3.45 Nucleophilic sub- 3-HBA (32) + pyruvate (7) chorismatases hygroscopicus stitution (arene subsp. oxide intermediate) hygroscopicus Type-III XanB2 (Q3I352) Xanthomonas 4.1.3.40/ Nucleophilic sub- 3-HBA (32)/4-HBA (6)+ chorismatases campestris pv. 45 stitution (arene pyruvate (7) campestris oxide intermediate) Type-IV SmCH-IV Stenotrophomonas 4.1.3.40 Nucleophilic sub- 4-HBA (6) + pyruvate (7) chorismatases (WP_051010542.1/ maltophilia stitution (arene NCBI ID) oxide intermediate)

Pericyclic Reactions).8,21,29,53 Members of this family are wide- an ‘ideal’ environment for product formation without an spread in bacteria, plants, and fungi, and provide important immediate catalytic contribution.8,21,29 intermediates for the biosynthesis of key primary metabolites (Iso)chorismate mutases convert (iso)chorismate (2/1) into such as tyrosine, phenylalanine (chorismate mutases), and (iso)prephenate (9/5) by a [3,3]-sigmatropic rearrangement ubiquinone (chorismate lyases) (Fig. 2).9,10,34 Beside the pri- (Claisen rearrangement), analogous to the non-enzymatic reac- mary metabolites, SPR enzymes catalyse the formation of tions (Scheme 1).54 The formation of isoprephenate (9) is so far salicylate, and other bioactive secondary metabolites such as only predicted based on the detection of the product in vivo;55 pyochelin (isochorismate-pyruvate lyases).8,12 All members of therefore, it is not clear if dedicated isochorismate mutases the SPR family share two unique features: (i) no intermediates exist. Studies on the biosynthesis of the frequently used anti- are formed during the reaction, and (ii) the active site provides biotic chloramphenicol (22) indicate that one intermediate

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Scheme 2 Molecular mechanisms of MST enzymes. (A) SN2-type mechanism including diol-carbanion intermediate from chorismate (1)to

Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. isochorismate (2) catalysed by isochorismate synthases. (B) SN2-type mechanism including hydroxylamine-carbanion intermediate from chorismate (1) to ADIC (3) catalysed by ADIC synthases. (C) SN2-type mechanism including diamine-carbanion intermediate from chorismate (1) via ADIC as an

intermediate (3)toADC(4) catalysed by ADC synthases. (D) SN2-type mechanism including enzyme-bound intermediate from chorismate (1) to ADC (4)

of ADC synthases. (E) Lyase reaction of isochorismate (2, X = OH) or ADIC (3,X=NH2) to salicylate (8, X = OH) or anthranilate (10,X=NH2), respectively, and pyruvate (7) including near attack conformation (NAC) and transition state (TS).

towards the route to the final product is 4-amino-4- usually catalysed by pyridoxal phosphate dependent para- deoxyprephenate (23) provided by a chorismate mutase-like aminobenzoate synthases (Scheme 1E).61 enzyme (ADC mutase),56,57 experimental proof is to our knowledge outstanding. (Iso)chorismate-(pyruvate) lyases catalyse the aromatisation of (iso)chorismate to 4-HBA (6) and salicylate (8) via a 3.1. Chorismate mutases and isochorismate-pyruvate lyases [1,5]-sigmatropic rearrangement with elimination of pyruvate (7, catalyse diﬀerent sigmatropic rearrangements in highly similar Scheme 1).18,19,21,53 In addition to the native reaction, isocho- active sites rismate-pyruvate lyases show a chorismate mutase side activity For chorismate mutases two diﬀerent enzyme folds, the with the non-physiological substrate chorismate, albeit with a AroH-type and the AroQ-type chorismate mutases, are known, substantially lower reaction rate.58–60 Other studies explain this both performing the formation of prephenate. Although they chorismate mutase side activity with co-purification of a naturely share no clear sequence homology (below 20% identity),29,62–64 48 occurring chorismate mutase from the heterologous host E. coli. the catalytic efficiency (kcat/KM) lies within one order of magni- The corresponding side reaction for wild-type chorismate mutases tude (0.95 105 to 8.00 105 M1 s1)29,62–64 and seems to be is not known. Additionally, chorismate lyases perform a non- achieved by a highly similar active site architecture.29 The natural, cofactor-independent elimination reaction of ADC (4) molar enthalpy of the reaction from chorismate to prephenate 1 yielding 4-aminobenzoate (24, pABA) and pyruvate (7), which is was determined as DrHm ¼ð55:4 0:23Þ kJ mol . In context

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site. These residues form an electrostatic gradient over the active site that promotes product formation.19,21,29,43,59 The active site residues involved in development of the electrostatic gradient are highly conserved: among them are two positively charged residues (2 Arg in AroH-chorismate mutases, Arg and Lys in AroQ-chorismate mutases and isochorismate-pyruvate lyases) that coordinate the C20-methylene group of the enoyl side chain over C1 of the cyclohexadiene ring by electrostatic interactions with the carboxyl group of the enoyl side chain (Fig. 4A, B, and D).19,21 Rearrangement yields prephenate (5) when the not-hybridised p-orbitals of the sp2 carbons overlap, and subsequently the new C–C bond is formed. In a concerted but highly asynchronous reaction, the C–O bond between C3 of the cyclohexadiene ring and the enol ether is broken (Scheme 1A).19 Formation of salicylate (8) from isochorismate (2) is catalysed in a similar fashion to prephenate (5) formation: here, instead of a C–C bond formation, a hydride shift from the sp2 carbon at C2 of the cyclohexadiene ring to the anti-parallel not-hybridised p-orbitals of the sp2 carbon at the C20-methylene group of the enoyl side chain occurs. In a concerted, asynchronous reaction, the C–O bond between C3 of the cyclohexadiene ring and the enol ether is broken during pyruvate formation 8,21,59,66

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. and aromatisation yielding salicylate (Scheme 1D). In chorismate mutases the C4-hydroxyl group of the cyclohexadiene ring is additionally stabilised by a highly conserved glutamate residue, while isochorismate-pyruvate lyases lack this residue. The resulting lower density of the electrostatic network in isochorismate-pyruvate lyases is assumed to be the reason for the promiscuity of this subfamily in contrast to chorismate mutases (Fig. 4A and B). The importance of the three amino acid residues for catalysis was highlighted by This article is licensed under a mutagenetic experiments with isosteric amino acid exchanges which resulted in inactive proteins or substantially lower Scheme 3 Suggested molecular mechanisms of isochorismatases and activity.68,69 The pericyclic nature of the isochorismate- type-I chorismatases. (A) Vinyl ether hydrolysis of isochorismate (2) pyruvate lyase reaction was shown for PchB by deuterium Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. and ADIC (3) to 2,3-trans-CHD (25) and 2,3-trans-CHA (26), respectively, labelling and subsequent NMR analysis as well as molecular and pyruvate (7) via a carbocation/oxocarbonium ion and an unstable 21 hemiketal intermediate of isochorismatases. (B) Vinyl ether hydrolysis of dynamics (MD) simulations. The formation of prephenate (5) chorismate (1) to 3,4-trans-CHD (27) and pyruvate (7) via the same starting from the non-native substrate chorismate (1)is mechanism proposed for chorismatases. catalysed via a comparable conformation, as shown for the native substrate isochorismate (Scheme 2D).58,67

with energy calculations and an equilibrium constant of 3.2. The pericyclic nature of the reaction and the conservation K E 7 109 the reaction can be considered as of the active site make chorismate mutases an ideal model irreversible.65 AroH-type chorismate mutases, such as the system to study enzymatic reactions chorismate mutase from B. subtilis,showapseudo-a/b-barrel Based on the wealth of experimental data, including three- structure with a three-domain architecture (Fig. 3D). This dimensional structures, site-directed mutagenesis, and label- results in three active sites at the interfaces between the ling studies, the pericyclic nature of the chorismate mutase single domains (Fig. 4D).29 AroQ-typechorismatemutases, reaction provides an almost ideal system to study this rare e.g. the E. coli chorismate mutase,62–64 share an all a-helical enzymatic reaction type theoretically.18,19,21,25,26,29,30,42,54,70 enzyme fold with isochorismate-pyruvate lyases along with a Such studies are based on QM/MM calculations that low sequence homology of roughly 20% (Fig. 3A and B). The often compare the sigmatropic rearrangements of chorismate active sites between the C- and the N-terminus of the single (1) in solution, the gas phase, and the enzymatic a-helical strands are highly similar (Fig. 4A and B).62–64,66,67 environment.22,25,26,30 Depending on the models used for these In SPR enzymes the chemical structure of the product calculations, diﬀerent states along the reaction trajectory are formed is controlled by an intricate network of mainly posi- recognised as being important for product formation, e.g. tively and negatively charged amino acid residues in the active the near attack conformation, the transition state, and the

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Scheme 4 1,4-Conjugate addition of SEPHCHC synthases. ThDP forms intermediate I after addition at C2 of 2-oxoglutarate (30) and decarboxylation of the C1 carboxyl group. Subsequent addition of isochorismate (2) leads to intermediate II before SEPHCHC (29) is released with regeneration of ThDP. Figure adapted from ref. 105

over the active site, represents the determining factor for This article is licensed under a catalysis.29,54,74,75 Recently, the detailed theoretical and experimental data available for chorismate mutases enabled Russ et al. to succeed

Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. in the rational design of artificial enzyme variants with nature- like catalytic activity in vivo.76 These active chorismate mutase variants have remarkable sequence diversity and were predicted by a sequence-based approach using evolutionary data. The results indicate that such statistical models may be of general usefulness for the design of artificial proteins with novel chemical activities.76

3.3. Chorismate lyases have a unique enzyme fold and catalyse a [1,5]-sigmatropic rearrangement Chorismate lyases such as UbiC from E. coli catalyse the [1,5]- Scheme 5 Modified E1cb mechanism of chorismate dehydratases. The carboxylate of the enolpyruvyl tail deprotonates chorismate (1)atC3ofthe sigmatropic elimination reaction of chorismate (1) yielding cyclohexadiene ring. Subsequently, the C4-hydroxyl group serves as a pyruvate (7) and 4-HBA (6, Scheme 1B).43 The chorismate lyase leaving group, with aromatisation of the cyclohexadiene ring leading to from M. tuberculosis is structurally conserved despite 12% 3-enoylpyruvyl benzoate (31). sequence homology.77 Chorismate lyases are an exception among chorismate-converting enzymes, since they are known to suﬀer a strong and competitive product inhibition, that is pseudo-diaxial conformation of chorismate in the active supposed to stabilise the protein fold.78 The three-dimensional site.25,29,30,54,71–73 The contribution of these effects has been structure has an anti-parallel six-stranded b-sheet covered by actively debated for the last two decades. Recent studies sup- two wing-shaped helix-turn-helix motifs forming the active site port the assumption that the electrostatic gradient, spanned all (Fig. 3C). In addition, these two ‘wings’ are expected to be

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Scheme 7 Anomeric eﬀect in chorismate derivatives. The free orbitals of the C4–oxygen of chorismate (1) cause an anomeric effect that weakens the C–O bond at C3 promoting bond breakage and product formation. An additional methyl group at the C4–oxygen increases this effect in 4-O-methylchorismate while the effect is absent in 4-deoxychorismate. This highlights the importance of the C4-hydroxyl group for product formation in chorismate-converting enzymes in general.

involved in product release. Another unique feature is the presence of distinct substrate and product binding sites in chorismate lyases.43,53 In contrast to isochorismate-pyruvate lyases, chorismate lyases are specific for their native reaction.43 The molecular mechanism is assumed to proceed analogous to the one described for isochorismate-pyruvate lyases. Chor-

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. ismate (1) is transferred from the substrate to the product binding site, in which the enolpyruvyl tail sterically hinders the stabilisation of chorismate (Fig. 4C). The C20-methylene group of the enoyl side chain is coordinated over C5 of the cyclohexadiene in a near attack conformation that develops in a pseudo-diaxial chair-like transition state.43,53 A hydride shift from the sp2 carbon at C4 of the cyclohexadiene ring to the anti-parallel not-hybridised p-orbitals of the sp2 carbon at the C20-methylene group of the enoyl side chain causes a C–O bond This article is licensed under a cleavage at C3. This results in the formation of pyruvate (7), as well as aromatisation yielding 4-HBA (6, Scheme 1B).43 The same overall reaction is also catalysed by type-III/IV choris-

Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. matases (see Section 8).

4. MST enzymes perform nucleophilic substitutions and lyase reactions in one enzyme fold

The enzymes involved in the biosynthesis of Menaquinone, Siderophores, and Tryptophan (MST enzyme family) catalyse the conversion of chorismate (1) into either isochorismate (2), ADIC (3), ADC (4), anthranilate (10), or salicylate (8, Scheme 2).7,8 Anthranilate synthases and ADC synthases, the most abundant MST enzymes, are involved in important pathways of primary metabolism.4,48,79 Salicylate synthases and isochorismate synthases play key roles in the biosynthesis of the electron carrier menaquinone and secondary metabolites, 80,81 Scheme 6 Putative molecular mechanisms of type-II, -III, and -IV choris- such as the siderophore enterobactin (13). The plant hor- matases. Starting from chorismate (1), a carbocation/oxocarbonium ion is mone salicylate plays a key role in plants’ defence against formed, followed by formation of an alcoholate at C4 and subsequent various threats.82–84 Only two ADIC synthases are known to attack at C3 leading to an arene oxide intermediate with concomitant loss date. This subfamily of MST enzymes is involved in the bio- of pyruvate (7). The ring opening via an NIH shift determines the products synthesis of phenazines that were shown to have potent anti- 3-HBA (32) and/or 4-HBA (6) depending on the chorismatase subfamily. biotic properties.85 All enzymes of the MST family perform a

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00 93 regio- and stereoselective nucleophilic substitution (SN2 ) position (Scheme 2C and D). Activation of the C4-hydroxyl resulting in an isomerisation and/or an amination depending group at the cyclohexadiene ring is catalysed by a conserved on the enzyme and the nucleophile. In the case of anthranilate glutamate via protonation and thus the formation of water as synthase and salicylate synthase, an additional lyase reaction is the leaving group. The corresponding activation of the nucleo- catalysed leading to the corresponding aromatised products phile is achieved via either a basic (Lys) or polar (Asn, Gln) with loss of pyruvate (7).7,8 Whether the nucleophilic substitu- residue. Basic residues are found in MST subfamilies where tion reaction of isochorismate synthases is reversible or irre- water serves as a nucleophile (isochorismate synthases, salicy- versible has been controversially debated for decades, based on late synthases). Polar residues are found in MST subfamilies contradicting experimental results from studies on the iso- where ammonia serves as a nucleophile (Asn – ADIC synthases; chorismate synthases EntC and MenF.9,86 Gln – ADC synthases, anthranilate synthases). Water leaves the molecule and causes a shift of the cyclohexadiene ring C–C 4.1. An homologous three-dimensional structure with a double bonds and a formal inversion of the stereochemistry highly conserved active site regarding the respective hydroxyl or amino moieties attached to The overall structure of MST enzymes is dominated by two C2 and C4 (Scheme 2) of the product compared to the orthogonal b-sheets decorated with a-helices and loops that substrate.7,8,48,92–94 In the case of ADC synthases, an alternative

determine the final a/b/b/a-fold (Fig. 3G). The active site is SN2-type substitution is catalysed where ammonia attacks at the located at the C-terminal part of the enzyme structure, C4 position of the intermediate described above and causes a embedded between the b-sheets. MST enzymes feature a retention of the stereochemistry relative to chorismate (1) sophisticated active site with contribution of at least 16 amino (Scheme 2C). Currently, the molecular mechanism of ADC acid residues. Nine of these are conserved over all MST synthases remains poorly understood and highly debated due enzymes. Three residues slightly diﬀer in their chemical prop- to the low quality of available structural data and contradicting erties, e.g. steric hindrance (Thr vs. Ser), acidic instead of polar results of studies on the molecular mechanism.4,87,95

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. (Asn vs. Asp/Glu), or different aromatic side chains (Trp/Phe vs. Tyr). The remaining four amino acids are characteristic for the 4.3. Enigmatic distinction between lyase activity and lyase individual MST subfamilies.79–81,87–90 In addition to the sub- deficiency in MST enzymes strate chorismate, a magnesium ion is bound in the active site The lyase reaction of salicylate and anthranilate synthases is via four acidic amino acid residues and the substrate’s C1 performed in an ordered-sequential mechanism. The long- 7,91 carboxyl group (Fig. 4G). A recent study by Meneely et al. suggested pericyclic nature of the reaction has been called into established the importance of this magnesium ion for catalysis doubt by Ziebart and Toney as well as by Culbertson et al. who 90–94 in isochorismate synthases and salicylate synthases. This is argue for an acid–base mechanism.48,96 In this case, the active most likely of global impact for MST enzymes, especially for site would promote a conformation of the intermediate iso- This article is licensed under a lyase-active enzymes in which the magnesium ion promotes the chorismate (2)/ADIC (3) that enables elimination of the enolpyruvyl progress on the reaction trajectory by favouring the formation side chain at C3 and aromatisation of the cyclohexadiene ring of reverse protonation states at the catalytic residues glutamate (Scheme 2E).8,48,81,91,96 So far, no clear hits for residues that and lysine. This is highlighted by the fact that lyase-deficient 7,48,92,96 Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. determine lyase activity or deficiency could be obtained. isochorismate synthases are inhibited by high concentrations A more detailed picture of these studies is given in the recent review 91 of magnesium, while lyase-active salicylate synthases are not. by Shelton and Lamb on MST enzymes.7 How exactly the formation of such a variety of products is accomplished in a single enzyme fold with an almost conserved active site is still a focus of research on chorismate-converting 5. Isochorismatases and type-I enzymes (see Section 8 to 10). chorismatases (chorismate 4.2. MST enzymes carry out regio- and stereoselective hydrolases): a typical hydrolysis nucleophilic substitutions reaction MST enzymes catalyse at least one SN2-type nucleophilic substitution utilising an acid–base mechanism.7,8,48 The substrate Isochorismatases catalyse the hydrolysis of isochorismate (2)to chorismate is protonated at the C4-hydroxyl group while an 2,3-trans-dihydroxycyclohexa-4,6-diene-1-carboxylate (25,2,3-trans- activated nucleophile (water or ammonia) attacks in a CHD) and pyruvate (7), as well as the hydrolysis of the si-fashion at C2 of the cyclohexadiene ring. This results in the corresponding amino derivative ADIC (3)yielding2,3-trans- formation of a diol/hydroxylamine-carbanion intermediate.7,48 dihydro-3-hydroxyanthranilate (26,2,3-trans-CHA).44,97 Beside In ADC synthases, the nature and genesis of this intermediate their physiological reaction, isochorismatases accept chorismate is discussed to be more diverse: ADC synthases such as PabB-II (1)and/orADC(4) as non-native substrates with substantially from S. maltophilia use ADIC (3) as an intermediate;95 for other lower catalytic efficiency (difference: 104).44,98,99 representatives of ADC synthases such as PabB-I from E. coli Chorismatases are one of the most recently discovered experimental data indicate an enzyme bound intermediate families of chorismate-converting enzymes. In total, four cho- between the catalytic lysine residue and the chorismate C2 rismatase subfamilies catalyse the conversion of chorismate (1)

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into (dihydro)hydroxybenzoates and pyruvate (7,Scheme3A).37,50 (30, a-ketoglutarate) yielding SEPHCHC, which can be Type-I chorismatases such as FkbO from S. hygroscopicus subsp. described as a Stetter-like 1,4-conjugate addition.45 The reac- ascomyceticus catalyse the conversion of chorismate (1)into3,4- tion product 29 is an intermediate in the biosynthesis of trans-dihydroxycyclohexa-1,5-diene-1-carboxylate (27,3,4-trans- menaquinone (vitamin K), an essential electron carrier during CHD) and pyruvate (7), but do not accept isochorismate (2)asa anaerobic growth in facultative anaerobic bacteria, e.g. substrate (Scheme 3B).27,47,100 E. coli.6,45 SEPHCHC synthases require thiamine diphosphate (ThDP) as a cofactor and elucidation of different three- 5.1. A superfamily determining fold and a hydrophobic active dimensional structures has indicated that MenD enzymes are site pocket magnesium (as in MST enzymes Section 4) or manganese Isochorismatases have the characteristic a/b-fold of the iso- dependent.91,102,103 Product formation is suggested to proceed chorismatase hydrolase superfamily but lack the catalytic triad via two sequential half-reactions including two covalent inter- that is common to most members of this superfamily.44,98,99,101 mediates (Scheme 4).104,105 The structure is characterised by a central six-stranded b-sheet 6.1. SEPHCHC synthases exhibit a typical ThDP-dependent that is covered on one side by three a-helices and a loop; this enzyme fold also defines the active site. On the opposite side, usually one large a-helix is spanned over the central sheet The three-dimensional structure of SEPHCHC synthases displays (Fig. 3F).44,98,99,101 In the active site, isochorismatases feature a three-domain architecture characteristic for ThDP-dependent a hydrophobic pocket stabilising the non-substituted side of enzymes. Domains I and III share the same topology featuring a the cyclohexadiene ring (Ile, Val, Leu, 2 Phe, 2 Tyr, Trp). central six-stranded b-sheet sandwiched between a-helices; A positively charged (Arg) and a polar (Gln) residue coordinate domain II shows a five-stranded b-sheet flanked by six the carboxyl group at C1 while the enolpyruvyl carboxylate is a-helices. The ThDP binding site, as well as the active site, is held in place by backbone interactions (Fig. 4F).44,98,99 The formed by two subunits of a homodimer at the interface, with

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. presence of an aliphatic amino acid residue (with a variety of contribution of the domains I and III of the different monomers side chains resulting in different steric hindrance) is assumed (Fig. 3I).102,103,105 Recently, the function of domain II to be responsible for the extent of substrate promiscuity of was elucidated by identification of an allosteric binding site isochorismatases, as it is the only variable residue across the for 1,4-dihydroxy-2-naphthoic acid, a downstream metabolite of active site.44,98,99 The chorismatase enzyme fold is unrelated menaquinone biosynthesis.106,107 and is discussed in Section 8.1.47,100 In addition to mostly hydrophobic amino acids contributing to the active site, usually five basic amino acid residues provide 5.2. An uncommon vinyl ether hydrolysis a suitable chemical environment to promote 1,4-conjugate 104,105,107–109 Isochorismatases and type-I chorismatases perform their reac- addition and stabilisation of the product. The most This article is licensed under a tions with fundamentally different enzyme folds and active site important residues that contribute to the process are: (i) a architectures that are highly conserved within the respective highly conserved glutamate activating ThDP, (ii) two conserved enzyme (sub)family.44,47,98–100 In both suggested mechanisms, arginines that are suggested to be crucial for substrate binding 0 and catalysis, and (iii) two polar amino acids (Gln and Ser) that Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. an acid protonates the C2 -methylene moiety of the enoyl side 103,105,107–109 chain with development of a carbocation/oxocarbonium ion at finally enable product formation (Fig. 4I). the C30 position. This is subsequently nucleophilically attacked 6.2. Impressive reaction sequence of the Stetter-like 1,4- by an activated water molecule in order to form an unstable conjugate addition of SEPHCHC synthases hemiketal intermediate. This hemiketal decomposes sponta- neously into either 3,4-trans-CHD (27) or 2,3-trans-CHD/CHA In SEPHCHC synthases, isochorismate (2) and 2-oxoglutarate (30) (25/26) and pyruvate depending on the substrate and the are ThDP-dependently linked in two sequential half-reactions with 105 enzyme (Scheme 3).44,47,97 3,4-trans-CHA (28) has also metastable intermediates (Scheme 4). The catalytic cycle of been reported as a product accessible from this hydrolysis MenD requires as an initial step activation of the cofactor ThDP. in vivo.31 The 4-aminopyrimidine ring of ThDP is transformed to the corresponding pyrimidinium and imino tautomers stabilised by the protein environment. These tautomers contribute to the formation 6. SEPHCHC synthases: a thiamine of a highly nucleophilic ylide species of the ThDP thiazole ring. C2 diphosphate dependent C–C coupling of the thiazole ring nucleophilically attacks 2-oxoglutarate to form reaction the post-decarboxylation intermediate I. Mutagenesis as well as structural and kinetic studies indicate that intermediate I is solely In contrast to other chorismate-converting enzymes which present in its carbanion or the protonated C2a-hydroxyalkyl deri- provide rearranged or cleaved products starting from (iso)cho- vative form. This is in contrast to other ThDP-dependent enzymes rismate or the corresponding amino derivatives, 2-succinyl-5- where the enamine form can be detected as well.102–105,107–109 ThDP enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylic acid (29, and the C2a-decarboxylated 2-oxoglutarate are proposed to be SEPHCHC) synthases, such as MenD from E. coli, catalyse a connected in a unique rigid conformation with a defined C–C coupling reaction of isochorismate (2) and 2-oxoglutarate stereochemistry by two conserved arginine residues keeping

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intermediate I in the proper orientation for nucleophilic attack 8. Chorismatases: one fold, two at isochorismate, which proceeds with formation of a novel C–C mechanisms, three products, four single bond, culminating in intermediate II.105,107–109 Finally, the interplay of two polar amino acids (Gln and Ser), the isochorismate subfamilies moiety, and the C2a-hydroxyl group of intermediate II, as well as As noted in Section 5 chorismatases can be classified into the aminopyrimidine ring of ThDP, in a complex hydrogen-bond several subfamilies. In addition to the hydrolysis reaction of network enables product formation. The aminopyrimidine ring type-I chorismatases, type-II, -III, and -IV chorismatases per- of ThDP abstracts a proton from the C2a-hydroxyl group that form a lyase reaction leading to aromatisation of chorismate induces bond breakage between C2 of the ThDP thiazole ring with release of pyruvate. Type-II chorismatases, such as Hyg5 and C2a of intermediate II, leading to regeneration of the from S. hygroscopicus subsp. hygroscopicus, catalyse the for- cofactor ThDP and formation of the product SEPHCHC (29, mation of 3-HBA (32).37,47,111 Type-III chorismatases, such as 103–105,107–109 Scheme 4). XanB2 from X. campestris pv. campestris, yield 3-HBA (32) and 4-HBA (6).112–114 Type-IV chorismatases, such as SmCH-IV from S. maltophilia, produce exclusively 4-HBA (6) from chorismate without competitive product inhibition as shown for choris- 7. Chorismate dehydratases provide a mate lyases, and with comparable catalytic efficiency.50,53 formal syn-elimination of water 8.1. A conserved enzyme fold with homology to AroH- Chorismate dehydratases are the most recent addition to the chorismate mutases chorismate-converting enzyme superfamily.46,110 MqnA catalyses Remarkably, the diﬀerent reactions of chorismatases type I, -II, the first step of an alternative biosynthetic pathway to menaquinone -III, and -IV forming various products are catalysed by a single via aminofutalosine. Genome network analyses indicate that chorismatase enzyme fold with an average 35% similarity on

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. chorismate dehydratases are involved in the biosynthesis of amino acid basis.47,50 This enzyme fold has a three-domain further natural products.6 The only structurally and mechanistically architecture displaying a pseudo-a/b-barrel structure with characterised member to date is MqnA from D. radiodurans;the homology to the RidA/YjgF superfamily, and, interestingly, formation of the product 3-enoylpyruvyl benzoate (31) from AroH-chorismate mutases (Fig. 3D and E). In contrast to the chorismate was first shown for MqnA from Thermus thermophilus trimeric AroH-chorismate mutases, chorismatases are mono- HB8.46,110 meric containing three similar domains. They have one active site with contribution of amino acid residues from the central

7.1. Alternative E1cb mechanism of chorismate dehydratases and C-terminal domains. A similar architecture, although with

This article is licensed under a the active site located in the center of the molecule, has been MqnAfollowsavariationofanE1cb mechanism in which substrate-mediated deprotonation is slower than loss of described for the cyanuric acid hydrolase AtzD, and coined 115 water.110 The structure of MqnA was solved with and without ‘‘Toblerone fold’’. The location of the active site was eluci- 3-HBA (32) as a ligand and product mimic. Detailed informa- dated by co-crystallisation of a competitive inhibitor (Fig. 4E): Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. tion about the overall three-dimensional fold was recently this structure suggests that chorismatases require chorismate presented as protein data base entries (Fig. 3H).46 The trans to be bound in a pseudo-diaxial conformation for 47,100,116 periplanar orientation of the substrate chorismate prevents catalysis. Chorismatases coordinate chorismate via a an E2 mechanism. However, a typical E1 mechanism would positively charged (Arg) and an aromatic (Tyr) residue at the require activation of the leaving group, which is only likely enolpyruvyl carboxylate. The carboxyl group at C1 is coordi- under acidic or neutral active site conditions. Such condi- nated by an arginine. Further, the substrate is stabilised via p–p tions were ruled out by the pH profile of MqnA. The putative interaction of the cyclohexadiene ring and a phenylalanine molecular mechanism of chorismate dehydration starts with residue. An acidic residue (Glu) is essential for at least one 47,100 an initial slow deprotonation at C3 of the cyclohexadiene ring crucial (de)protonation step during catalysis. with formation of a carbanion. The carboxylate of the enolpyruvyl tail is suggested to act as the base for deprotonation, 8.2. The lyase reaction of chorismatases involves an as there is no acidic or basic amino acid residue found in the unexpected arene oxide intermediate active site with bound 3-HBA that could assume this role. The Type-I (FkbO-like) chorismatases catalyse a vinyl ether hydro- carbanion is suggested to be stabilised via delocalisation lysis as described for isochorismatases (see Section 5.2). In over the C–C double bonds of the cyclohexadiene ring and contrast to type-I chorismatases, the lyase-active type-II, -III, the carboxylate at C1. The C4-hydroxyl moiety of the cyclo- and -IV chorismatases use a fundamentally diﬀerent catalysis hexadiene ring serves as the leaving group and exits the (Scheme 6). The proposed molecular mechanisms are predo- molecule relatively quickly compared to the other reaction minantly based on labelling experiments using selectively steps (Scheme 5).46 Thebackboneamidegroupofaserine labelled substrates and labelled solvents.47,50 A few active residue is assumed to contribute via stabilisation of the site residues in conserved amino acid motifs determine the leaving group (Fig. 4H).46 mechanism and the product(s) formed. A carbocation at the

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C30 position is formed through initial protonation at the of chorismate where the coordinating cysteine residue has no C20-methylene group of the enoyl side chain that is common influence on ring opening and the ring opens to the favoured to all four chorismatase subfamilies. In type-II to -IV chorisma- product 4-HBA. Ala and Asp at the respective positions locate tases, deprotonation of the C4-hydroxyl group at the cyclohex- chorismate between the two positions in the active site that is adiene ring is suggested to be the next crucial step during linked to a loss of product selectivity.47,50,117 catalysis, carried out by the same catalytic glutamate used before for protonation. The assumed C4-alcoholate is suggested 8.4. Chorismatases are a starting point for the discovery of to perform an intramolecular nucleophilic attack at C3 of the novel natural products with rare meta-substituted moieties cyclohexadiene ring resulting in the formation of an arene Chorismatases are involved in the biosynthesis of a wide range of oxide intermediate, with pyruvate as a leaving group. The arene bioactive natural products. Type-I chorismatases provide the starter oxide intermediate is opened to yield 3- and/or 4-HBA (32/6) unit for the biosynthesis of immunosuppressants such as rapamycin determined by the active site architecture of the respective (19,Fig.2)andascomycin.37 Type-II and -III chorismatases are chorismatase subfamily. The arene oxide opening is proposed involved in the biosynthesis of unusual meta-substituted natural to be linked to a concerted NIH shift, characterised by migra- products such as the polyketidic BC325 (a naturally occurring tion of the proton at the position of the product hydroxyl group rapamycin derivative), cuevaene A (33), xanthomonadin-dialkyl at the cyclohexadiene ring (C3 – 3-HBA, C4 – 4-HBA) to the ring resorcinol and xanthomonadin I (34), and the terpene brasilicardin position where the C–O bond of the arene oxide intermediate (35).37,111–113,118 Only a few other meta-substituted natural products, was broken. This proton would finally be released during e.g. 3-formyl-tyrosine and pacidamycin, are known.119,120 Conse- 47,50,117 aromatisation of the ring. The fundamental different quently, type-II and -III chorismatases are promising handles to molecular mechanisms of chorismatases catalysed in highly detect novel biosynthetic pathways for compounds with this other- similar active sites of conserved enzyme folds make chorisma- wise rare structural feature. Finally, type-III and -IV chorismatases tases an interesting target for the theoretical calculation of catalyse the formation of 4-HBA (6) during the biosynthesis of 117 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. these reactions. These calculations support the proposed ubiquinones. All studied organisms containing a type-III or -IV molecular mechanisms and suggest further amino acid chorismatase lack a conventional chorismate lyase in their genome. residues outside of the active site that may play a key role in Therefore, type-III and -IV chorismatases are suggested to represent the selectivity for one distinct molecular mechanism (hydro- the starting point of an alternative route to ubiquinone in primary 24,50,117 lysis or arene oxide). metabolism.50,113 In addition, 4-HBA might serve as starter unit for Therespectiveaminoacidresidueswerealsodetectedasa the biosynthesis of novel arylpolyenes as the studied new type-IV correlation pair in bioinformatic analysis of the different chor- chorismatases are embedded in yet undescribed arylpolyene biosyn- ismatase types (see Section 8.3) but ruled out for a role in product thetic gene clusters.50 The ability to securely predict the products of 50,117 formation based on their distance to the active site. an unknown chorismatase is a useful tool for the prediction of novel This article is licensed under a biosynthetic pathways and was successfully used in the discovery of 8.3. Three correlating pairs of amino acid residues are 38,50 responsible for the product outcome of chorismatases unantimycins (e.g., 20). Correlation analysis of a set of (putative) chorismatase protein Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. sequences revealed a pattern of characteristic variable amino 9. Interchange or permute hypothesis? acid residues in the active site determining the molecular mechanism catalysed:47,50 in type-I chorismatases two non- As described in the previous sections, chorismate-converting polar residues (2 Ala) ensure the correct position of the enzymes of one specific (sub)family often share conserved substrate in the active site for the vinyl ether hydrolysis amino acid sequences and are structurally similar (at least) at reaction.50,100 In type-II, -III, and -IV chorismatases, these two the active site. The MST enzyme family is an outstanding residues are substituted by a conserved glycine and a conserved example of this: Meneely et al. investigated whether product cysteine residue. These substitutions are suggested to change selectivity, the molecular mechanism followed, and the choice the orientation of chorismate in the active site to enable of nucleophile in the MST enzyme family are determined by (i) deprotonation at the C4-hydroxyl group and concomitant for- single active site residues, or (ii) a more elaborate interplay of mation of the proposed arene oxide intermediate. The product active site residues and electrostatic networks.49 If hypothesis range of type-II, -III, and -IV chorismatases is putatively deter- (i) holds true, an interchange of the corresponding enzyme mined by the electrostatic repulsion and the steric require- family specific native reactions between different MST enzyme ments of an active site residue (Gly, Ala, or Cys) adjacent to the families would be feasible by site-directed mutagenesis (inter- C1 carboxylate of the cyclohexadiene ring, and a Glu or Asp change hypothesis). In the case of hypothesis (ii), non-native residue forming a hydrogen bond with the catalytic glutamate. reactions would be catalysed on a low level after mutagenesis, A combination of Gly and Glu brings the arene oxide inter- with reduced catalytic efficiency in addition to the physiological mediate in a position such that the ring opening is selective to reaction. After site-directed mutagenesis of specific active site provide the meta product 32 likely supported by electrostatic residues, the result would be (highly) promiscuous enzymes with a interaction with the Cys residue responsible for mechanistic significantlossofproductselectivity (permute hypothesis). The selectivity. A combination of Ala/Cys and Glu leads to a position interchange or permute hypothesis was tested by attempting to

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install a lyase activity in lyase-deficient MST enzymes, with the molecular mechanisms that selectively lead to a broad range outcome that the permute hypothesis is more likely.49 Afinalproof of products. The growing comprehension of the underlying for the permute hypothesis is diﬃcult, but most results using site- molecular mechanisms in combination with the three- directed mutagenesis support this finding.4,47,48,80,81,88–90,92,94 Never- dimensional structures have immediate impact on the broader theless, there are a few examples that support the interchange understanding about the whole group of chorismate-converting hypothesis as well, e.g. adoublemutantoftheADCsynthase enzymes, and might be transferrable to other enzyme families. PabB-I (Lys274Ala/Gln211Lys) that solely produces ADIC under The recent discovery of chorismatases and chorismate dehy- physiological conditions – without any detectable wild-type activity. dratases added to and shed new light on long held textbook However, all examples show a significant loss of catalytic activity, knowledge.37,50,110 Recent studies focused on either the MST or thereby raising the question of whether other products would be the SPR enzymes have contributed to this process as well.29,49,54,91 detected using increased reaction times.48,51,96 In summary, the In the following, we highlight a few general trends that seem suggestion that MST enzymes react to variations introduced in the important for the whole range of chorismate-converting enzymes. active site with significant promiscuity and loss of product selectivity is reasonable.49 10.1. Electrostatic interactions in the active site reflect the This trend is mirrored in other families of chorismate- reaction’s complexity converting enzymes. Pure interchange approaches in SPR Two global assumptions can be derived from studies on the enzymes aimed at establishing a chorismate mutase activity structural and mechanistic understanding of chorismate- in the isochorismate-pyruvate lyase PchB resulted in promiscu- converting enzymes: (i) a higher number of polar and charged ous variants and loss of activity.8,42,67,69,121,122 Similar results amino acid residues is observed in the active sites of enzyme were found when site-directed mutagenesis was applied to subfamilies catalysing more intricate mechanisms compared to interchange the diﬀerent molecular mechanisms of chorisma- enzymes with less sophisticated mechanisms of the same tases: promiscuous variants with median to dramatic loss of family, and (ii) the more complex the molecular mechanism, 47,50

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. catalytic activity were obtained. Nevertheless, this see- the higher the degree of promiscuity caused by mutagenesis. mingly undesired loss of selectivity and activity can be seen It is difficult to clearly define the level of complexity for the as a necessary first step on the way to a new activity, which is different molecular mechanisms. As a guideline we suggest (a) gradually introduced. Indeed, type-III chorismatases could be the higher the number of bonds broken and newly formed the regarded as a natively occurring promiscuous variant of lyase- more complex the mechanism, and (b) the lower the number of active chorismatases, as this subfamily leads to a mixture of products yielded the more complex the mechanism, particu- 3-HBA and 4-HBA.47,50,113 The ‘unselectivity’ of type-III chor- larly when a large number of products can theoretically be ismatases might thus be an evolutionary snapshot, represent- formed. In our opinion, these criteria allow to some extent a ing an intermediate step during evolutionary adaption of comparison of complexity between different chorismate- This article is licensed under a type-IV towards type-II chorismatases or vice versa. A similar converting enzyme families. As an example, a pericyclic Claisen assumption was made for the evolution from anthranilate rearrangement is considered to be more complex than a lyase synthases acting in the primary metabolism to isochorismate reaction (including re-aromatisation), which is again of higher synthases acting in the secondary metabolism by exchange of complexity than a hydrolysis. Examples of an increased number Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. two amino acids instead of gene duplication.51 of polar and charged residues in the active sites of (iso) In conclusion, the interchange experiments performed for chorismate-converting enzymes catalysing such sophisticated different families of chorismate-converting enzymes suggest that molecular mechanisms are summarised in the following. distinct chorismate-converting enzyme subfamilies have evolved to SPR enzyme family. Chorismate mutases catalyse the Claisen be selective for their native reaction. Regarding the reactivity of rearrangement of chorismate to prephenate, isochorismate-pyruvate chorismate and isochorismate, and the number of reactions pos- lyases perform a slightly less complicated reaction – [1,5]- sible, this requires a precise control of substrate binding and sigmatropic rearrangement yielding salicylate and pyruvate. We reaction. Therefore, it is not overly surprising that the evolution conclude this – although an equal number of bonds are broken from one activity to another cannot be easily copied with a few (3) and newly formed (3) – as only one product is formed in the point mutations leading to the same efficiency and selectivity. chorismate mutase reaction (isochorismate-pyruvate lyases yield two products). Moreover, the electrostatic network spanned over the active site of chorismate mutases is denser, meaning that more 10. Recently added enzyme families polar amino acid residues contribute to its formation compared to and mechanistic understanding isochorismate-pyruvate lyases.8,19,21,29,60 This was reinforced by the exchange of an arginine residue for the isosteric and non-charged provide unexplored mechanistic citrulline in chorismate mutase, that resulted in a dramatic perspectives for chorismate- reduction of electrostatic interaction.29 These experimental results converting enzymes are supported by theoretical calculations that suggest electrostatic stabilisation as the reaction’s rate-limiting factor. Nevertheless, As outlined above, chorismate-converting enzymes are a versa- additional QM/MM studies indicated a more entropy-driven reac- tile group of enzymes with fascinating and sophisticated tion, keeping the debate alive.

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MST enzyme family. The initial nucleophilic substitution in chorismate dehydratases: here, the C4-hydroxyl group is the all five subfamilies of the MST enzyme family can be con- leaving group during elimination of water and subsequent sidered of equal complexity. Consequently, the additional aromatisation.46 intermolecular nucleophilic substitution in ADC synthases is The influence of the C2/C4-hydroxyl group, especially in more complex compared to isochorismate synthases and ADIC lyase-catalysing chorismatases, indicates a significant impor- synthases. However, the product formation is less complex tance of this moiety for product formation in (iso)chorismate- compared to the lyase reaction of anthranilate synthases and converting enzymes: it is conceivable that the C2/C4-hydroxyl salicylate synthases, based on the number of bonds broken and group also essentially contributes to the molecular mecha- newly formed (4 vs. 6). In addition, the number of polar amino nisms of SPR enzymes as well as the lyase-active MST enzymes. acid residues that contribute to stabilisation of the C2-hydroxyl This contribution could be based on stereoelectronic eﬀects, group is higher the more complex the reaction: the lyase-active e.g. hyperconjugation or the anomeric eﬀect, or the development salicylate synthases have one more polar residue in the active of an arene oxide intermediate. The latter has been suggested site than the lyase-deficient isochorismate synthases.7 for lyase-catalysing chorismatases, as further outlined in the Chorismatases. In chorismatases, we judge the aromatisa- following. tions of type-II, -III, and -IV more complex than the hydrolysis- active type-I. In the lyase reactions an extra bond is broken 10.3. Is the arene oxide postulated for chorismatases also a (4 vs. 3) compared to type-I. Both reactions end up in the feasible intermediate for other reactions? formation of two products. In addition, a polar cysteine residue The recent discovery of chorismate dehydratases and choris- determines the lyase activity compared to an alanine residue in matases highlights that intramolecular reactions with con- hydrolase-active type-I chorismatases. Further, the exchange of tribution of acidic and/or basic active site residues are not a glutamate for a smaller aspartate is thought to disturb the unusual in chorismate-converting enzymes.46,47 This raises the electrostatic stabilisation of the essential catalytic glutamate in question of whether the pericyclic reactions in chorismate-

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. the rather unselective type-III chorismatases. This suggests that converting enzymes are just formally pericyclic, with true the localisation of the substrate in the electrostatic environ- pericyclic non-enzymatic counterparts in solution. Several ment of the active site determines the product selectivity. This results of studies on enzymatic and non-enzymatic reactions assumption is supported by experimental and theoretical of (iso)chorismate support this hypothesis. data.24,47,50 The pericyclic reactions catalysed by SPR enzymes, the lyase reaction of MST enzymes, and the reactions catalysed by 10.2. The C2/C4-hydroxyl group is important for product chorismatases require a pseudo-diaxial conformation of (iso)- formation chorismate, for which the enzymes are assumed to be From structural data and molecular modelling studies it is selective.24,25,29,30,54,71–75,117 In all these cases, the position of This article is licensed under a likely that the pseudo-diaxial conformation of chorismate the enolpyruvyl tail is essential for product formation and is a prerequisite for catalysis in chorismate-converting determines the molecular mechanism and the product selec- enzymes.7,18,19,21,29,44,46,47 Consequently, the C2/C4-hydroxyl tivity in a complex interplay with polar and/or charged amino group of isochorismate/chorismate, respectively, usually con- acid residues in the active site.7,19,21,47,100 Consequently, if the Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. tributes to some extent to the product formation (Scheme 7). enolpyruvyl side chain is of general importance for product In chorismatases, the molecular mechanisms can be inter- formation and (iso)chorismate has a pseudo-diaxial conforma- preted to resemble the limits of the C4-hydroxyl group’s influ- tion, the C2/C4-hydroxyl group as its counterpart is likewise ence on product formation. In type-I chorismatases catalysing a of general importance for product formation. Studies with vinyl ether hydrolysis there is a minor influence that is 4-deoxychorismate and 4-O-methylchorismate in solution sug- assumed to be limited to the stabilisation of the catalytically gest that an anomeric effect caused by the additional methyl active conformation.47,100 In the lyase-catalysing type-II, -III, group increases the reaction rate by weakening the C–O bond at and -IV chorismatases the C4-hydroxyl group is essential for C3 of chorismate more than the hydroxyl group usually does product formation as it attacks intramolecularly at the C3 (Scheme 7).123 Moreover, Hilvert et al. observed a difference position leading to the proposed arene oxide intermediate.47 between the theoretical calculations for the pericyclic reactions This situation is also found in other chorismate-converting of (iso)chorismate and the experimental results for the enzy- enzymes. In isochorismatases, the influence of the C2-hydroxyl matic but not for the non-enzymatic reactions.18,19,21 Finally, group on product formation is comparably low, as is the case the finding that the reaction proceeds in a highly asynchronous for the C4-hydroxyl group in type-I chorismatases.97,99 In but pericyclic manner seems to be a contradiction in itself. SEPHCHC synthases, the influence of the C2-hydroxyl group Consequently, these studies might serve as a hint that the C2/ seems to be minor as well.105,108 In MST enzymes, the C4-hydroxyl group is of general importance for catalysis.18,19,21 C2/C4-hydroxyl group has an immediate influence on product Although shown by kinetic isotope effects that the reactions formation as it serves as a leaving group for the nucleophilic proceed in a concerted fashion,18,19,21 the concerted nature of substitution while being (re)installed during the same reaction. the reaction does not exclude the formation of an elusive arene Its presence is therefore essential but diﬀerent to the situation oxide intermediate as suggested for type-II, -III, and -IV in the chorismatase subfamilies.7 The same might be true for chorismatases.47,50 In chorismate mutases, an alternative

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reaction mechanism with an arene oxide intermediate has been Allosteric regulation of chorismate-converting enzymes was suggested before.124 The conserved glutamate residue interact- first identified for chorismate mutases and their involvement in ing with the C4-hydroxyl group might serve as the base for the biosynthesis of aromatic amino acids. Accordingly, it was deprotonation and an additional cysteine residue would be shown that many chorismate mutases from various organisms are responsible for the faster opening of the arene oxide to the C4 allosterically regulated via feedback inhibition by the aromatic position. This cysteine residue would be an explanation for the amino acids Phe, Tyr, and Trp.128 Additionally, inter-enzyme higher catalytic efficiency in AroH-chorismate mutases com- allostery has been reported for chorismate mutase, e.g. in pared to AroQ-chorismate mutases that lack this residue.29,62 M. tuberculosis and Corynebacterium glutamicum where Trp-bound An arene oxide intermediate has been suggested also in the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase binds to the biosynthesis of salicylate.125 This idea can be transferred to other inactive chorismate mutase, with the resulting complex increasing lyase reactions of chorismate, as it was indirectly shown for chorismate mutase activity thereby directing additional flux into chorismatases that the formation of 4-HBA proceeds most likely the biosynthesis of aromatic amino acids.54,129,130 via such an intermediate.47,50 The introduction of deuterium into In MST enzymes, allosteric regulation has been described pyruvate from selectively deuterium-labelled isochorismate and for the glutamine amidotransferase domain of ADC synthases. the asynchronous nature of the isochorismate-pyruvate lyase Here, the formation of the nucleophile ammonia is allosteri- reaction support a pericyclic reaction as well as an arene oxide cally activated by chorismate, binding in the active site. This intermediate.21 Judging from the enzyme structures available, the concurs with the need of the nucleophile for catalysis.131 The activesiteenvironmentofisochorismate-pyruvate lyases, as well allosteric activation of the amidotransferase domain ensures as of salicylate synthases and anthranilate synthases, would be that glutamine is only consumed if chorismate is available for suitable to stabilise an arene oxide intermediate. An amino acid conversion into the corresponding amino derivative ADC. Trp residue that could serve as the base is also present in these abolishes catalysis in anthranilate synthases allosterically by enzymes.¶ 7,21,67 Moreover, structural data of salicylate and eﬀecting an ammonia channel to the active site without major 132

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. anthranilate synthases show that the enolpyruvyl side chain is structural changes. Additionally, structural data with bound embedded in a dense network of hydrogen bonds, keeping this Trp imply that Trp binding induces changes in the hydrogen- part of the molecule firmly in position during product binding patterns of anthranilate synthase hindering the opti- formation.7,81,96 This rigid network might actually hinder the mal formation of the active site.133 movementofthesidechainovertheringduringthelyase Recently, allosteric regulation of the SEPHCHC synthase MenD reaction, thus making a pericyclic reaction less likely. In chor- from M. tuberculosis was identified to be essential for the metabolic ismatases, the enolpyruvyl tail is suggested to assume a similar flux of menaquinone biosynthesis.106,107 The catalytic activity of position during formation of the arene oxide intermediate.47 MenD is inhibited by binding of 1,4-dihydroxy-2-naphthoic acid in In summary, an alternative reaction mechanism with formation an ‘arginine cage’ located at domain II of the enzyme. Binding of This article is licensed under a of an arene oxide intermediate for other chorismate-converting the inhibitor at the allosteric site causes structural changes that enzymes is imaginable but to date elusive. Experimental affect the position of a catalytically essential glutamate residue as approaches to support this hypothesis are challenging as the arene well as the flexibility of the active site. Nevertheless, a multi- oxide intermediate has to be scavenged by strong nucleophiles and/ sequence alignment of 35 MenD amino acid residues revealed that Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM. or detected spectroscopically.126 the three key arginine residues of the allosteric binding site show low conservation. This indicates that allosteric regulation of MenD 11. The metabolic flux into the by 1,4-dihydroxy-2-naphthoic acid is not a universal feature to direct metabolic flux in menaquinone biosynthesis.106,107 different branches starting from In conclusion, the identification of allosteric control in (iso)chorismate is directed by allosteric various chorismate-converting enzymes suggests that future regulation work in this direction might reveal such control to be a wide- spread feature to direct metabolic flux into the different (iso)- Chorismate is a metabolic node with many different branches; chorismate biosynthetic pathways. consequently, in each organism with a shikimate biosynthetic pathway multiple chorismate-converting enzymes are found.1 12. Chorismate-converting enzymes The metabolic flux into the specific branches plays a key role in biosynthesis of the corresponding (iso)chorismate derivatives. are important tools for In addition to straightforward mechanisms such as the product biotechnological and biocatalytic inhibition described for chorismate lyases (see SPR enzyme applications section), an important way to control this flux is allosteric regulation. The binding of an allosteric regulator distant from Chiral building blocks are often challenging to access by the active site can cause feedback inhibition or activation of the conventional organic synthesis. Enzymes in general are catalytic activity.127 emerging as biocatalysts for the selective chemoenzymatic

¶ Anthranilate synthases: Gln236/Thr425/His398 in TrpE-I, isochorismate-pyruvate lyases: Gln90 in PchB, salicylate synthases: Lys205/Thr361/Lys438 in MbtI.

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synthesis of chiral building blocks in vitro and in vivo.134,135 The metabolic branching point(s) of (iso)chorismate oﬀer access to many chiral and/or aromatic compounds.31 Several of these compounds have been made accessible on gram scale from rationally engineered E. coli strains, based on a growing understanding of metabolic flux of the biosynthetic pathways branching from (iso)chorismate.11,136,137 Microbial production of chemicals in vivo is complemented with the in vitro production of compounds such as isochorismate itself.27 In addition to such technical applications, chorismate- converting enzymes harbour a large potential as biocatalysts. An outstanding example among chorismate-converting enzymes are the SEPHCHC synthases. The promiscuity of Fig. 5 SEPHCHC (29) and derivatives 36–38 from biocatalytic applica- MenD makes this enzyme a versatile in vitro catalyst for the tions of wild-type MenD and variants thereof. asymmetric synthesis of numerous 2-hydroxy ketones by varia- tion of the donor and acceptor substrates.138,139 The non- physiological isochorismate derivatives SDHCHC (37) and MDHCHC (38) from the reactions of 2,3-trans-CHD (25) with 2-oxoglutarate or (S)-4-hydroxy-2-oxoglutarate, respectively, are accessible by wild-type MenD in vitro (Fig. 4). Recently, the substrate and product range of wild-type MenD was extended by (5S,6S)-6-amino-2-(3-carboxypropanoyl)-5-hydroxycyclohex-3-ene-1-

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. carboxylate (38, SAHCHC) that was detected in traces from the conversion of 2,3-trans-CHA (26) with 2-oxoglutarate (Fig. 5).31,32,139 Moreover, Fries et al. reported the preparative in vivo production of SEPHCHC, (iso)SDHCHC, and (iso)SAHCHC using engineered E. coli strains. Rationally designed MenD variants, based on a docking model with ‘1,6-dihydroisochorismate’ as a surrogate intermediate, increased the yields of fermentations and in vitro reactions significantly (6- and 23-fold). The occur- This article is licensed under a rence of some of these rationally designed variants in nature, detected by protein sequence network analysis, suggests that further chorismate-converting enzymes – in particular enzymes acting on the amino derivatives – await discovery.32 Open Access Article. Published on 19 February 2021. Downloaded 9/27/2021 8:25:03 PM.

13. Is there a hidden chorismate- Fig. 6 Natural products that are expected to be biosynthesised via a route converting enzyme potential encoded containing uncharacterised chorismate-converting enzymes: echinosporins (39) and tundrenone (40) of bacterial origin, the fungal natural in natural products? products chorismeron (41) and shikimeran B (42), and the plant metabolite glutamyl-isochorismate (43). In addition to the chorismate-converting enzymes characterised regarding their three-dimensional structures and molecular mechanisms, natural products provide insight into MenD.32,39,140 In addition, the elucidation of the tundre- the cryptic potential of enzymatic novelty by retro-biosynthetic none (40) biosynthetic pathway suggests an undescribed analysis and scavenging of intermediates. In light of this, it is C–C coupling reaction utilising chorismate as a substrate likely that further unprecedented enzyme families will be (Fig. 6).141 discovered.32,46,50 Reconsidering Fig. 1, the products accessible Higher organisms provide another putative source of from (iso)chorismate (1 and 2) by enzymatic and non-enzymatic unexplored chorismate-converting enzymes. Consequently, reactions are not fully covered for the corresponding amino co-cultivation of fungi with diﬀerent bacteria led to the identifi- derivatives ADIC (3) and ADC (4). Identification of such as yet cation followed by characterisation of structurally unique fun- enigmatic enzymes might lead to the amino derivatives being gal chorismate metabolites, e.g. chorismeron (41) and recognised as important metabolic branching points contribut- shikimeran B (42) (Fig. 5).142,143 Further, an unprecedented ing to a much larger biosynthetic diversity, as was proposed for chorismate derivative was detected in plants, where salicylate the biosynthesis of stravidins (18), echinosporins (39), and biosynthesis occurs via a three-enzyme cascade in two cell SEPHCHC derivatives (36–38) prepared with variants of compartments. Glutamyl-isochorismate (43) plays a key role

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by releasing the final natural product salicylate via a lyase 19 S. K. Wright, M. S. DeClue, A. Mandal, L. Lee, O. Wiest, reaction (Fig. 6).82–84 W. W. Cleland and D. Hilvert, J. Am. Chem. Soc., 2005, 127, 12957–12964. 20 K. A. Reynolds, K. K. Wallace, S. Handa, M. S. Brown, H. A. McArthur and H. G. Floss, J. Antibiot., 1997, 50, 701–703. 14. Conclusions 21 M. S. DeClue, K. K. Baldridge, D. E. Ku¨nzler, P. Kast and D. Hilvert, J. Am. Chem. Soc., 2005, 127, 15002–15003. The recent advances in the field of chorismate-converting 22 S. Hur and T. C. Bruice, J. Am. Chem. Soc., 2003, 125, 10540–10542. 23 S. Hur and T. C. Bruice, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, enzymes demonstrate the dynamic nature of this field. 12015–12020. Many enigmatic processes require further studies to enable a 24 Y. Zhang, H. Zhang and Q. Zheng, Chem. – Eur. J., 2019, 25, thorough understanding of such a unique metabolic node. In 1326–1336. 25 K. E. Ranaghan and A. J. Mulholland, Chem. Commun., 2004, addition, recent studies on biosynthetic pathways of natural 1238–1239. products and bioinformatic analysis directed towards the abun- 26 F. Claeyssens, K. E. Ranaghan, F. R. Manby, J. N. Harvey and dance of recently discovered chorismate-converting enzymes A. J. Mulholland, Chem. Commun., 2005, 5068–5070. 27 F. Hubrich, M. Mu¨ller and J. N. Andexer, J. Biotechnol., 2014, 191, indicate that there is a large and untapped enzymatic and 93–98. natural product potential. Therefore, we are convinced that 28 Y. B. Tewari, A. M. Davis, P. Reddy and R. N. Goldberg, J. Chem. this metabolic node will continue its action in the future. Thermodyn., 2000, 32, 1057–1070. 29 D. Burschowsky, A. van Eerde, M. Okvist, A. Kienho¨fer, P. Kast, D. Hilvert and U. Krengel, Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 17516–17521. Conflicts of interest 30 X. Zhang and T. C. Bruice, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 18356–18360. There are no conflicts to declare. 31 J. Bongaerts, S. Esser, V. Lorbach, L. Al-Momani, M. A. Mu¨ller, D. Franke, C. Grondal, A. Kurutsch, R. Bujnicki, R. Takors, L. Raeven, M. Wubbolts, R. Bovenberg, M. Nieger, M. Schu¨rmann, N. Trachtmann, S. Kozak, G. A. Sprenger and Acknowledgements M. Mu¨ller, Angew. Chem., Int. Ed., 2011, 50, 7781–7786. Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 32 A. Fries, L. S. Mazzaferro, B. Gru¨ning, P. Bisel, K. Stibal, We would like to thank Dr Jodie Johnston (University of Canter- P. C. F. Buchholz, J. Pleiss, G. A. Sprenger and M. Mu¨ller, ChemBioChem, 2019, 20, 1672–1677. bury, NZ), Prof. Georg A. Sprenger and Prof. Ju¨rgen Pleiss 33 C. T. Walsh, S. W. Haynes and B. D. Ames, Nat. Prod. Rep., 2012, 29, (University of Stuttgart, Germany), and Prof. Kai Tittmann 37–59. (University of Go¨ttingen, Germany) for many scientific and 34 V. Tzin and G. Galili, Mol. Plant, 2010, 3, 956–972. 35 F. Gibson and L. M. Jackman, Nature, 1963, 198, 388–389. philosophical discussions on chorismate-converting enzymes 36 A. R. Knaggs, Nat. Prod. Rep., 2003, 20, 119–136. in the last 10+ years; as well as Dr Jodie Johnston, Prof. Georg A. 37 J. N. Andexer, S. G. Kendrew, M. Nur-e-Alam, O. Lazos, T. A. Foster, Sprenger, and Dr Kay Greenfield (Brisbane, Australia) for proof- A.-S. Zimmermann, T. D. Warneck, D. Suthar, N. J. Coates, F. E. Koehn, J. S. Skotnicki, G. T. Carter, M. A. Gregory,

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